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https://hdl.handle.net/2144/15347

Abstract

Low-template deoxyribonucleic acid (DNA) samples are commonly found within forensic biological evidence. Low amounts of DNA become increasingly difficult to analyze as the allelic peaks become less distinguishable from instrumental noise. Forensic laboratories currently try to increase allele signal intensity through additional polymerase chain reaction (PCR) cycles or enhancing capillary electrophoresis injection times or potentials. Purification of the post-PCR product may also be conducted as PCR reagents can compete with DNA fragments during electrokinetic injection. Though these strategies have proven useful, resulting in a higher signal to noise ratio, low-template samples continue to exhibit allele drop-out due to the stochastic variation induced by the forensic DNA laboratory process. Further complicating analysis is the fact that low-template DNA samples are often exhausted as the full amount is needed for analysis. Thus, PCR can be considered a destructive technique. Since allele drop-out is hypothesized to be the result of 1) insufficient levels of amplicons and 2) sampling effects, it is desirable to obtain the original DNA template after amplification for future analysis. This would minimize the impact of 1) above.
Thus, a novel method which isolates genomic DNA after PCR amplification has been developed. Amplification products were produced using biotinylated primers and cleaned from the solution with streptavidin-coated magnetic beads. Filtration was then used to remove remaining PCR reagents and primers. The result is a recovered sample containing the original genomic DNA. Re-amplification was then performed showing the method is successful.
Although the method is capable of re-amplifying isolated DNA after PCR, there are points within the procedure that need to be optimized. For example, significant amounts of DNA are lost during the cleaning process and there is a high retention of the original amplified product. This study describes the optimization steps taken to reduce DNA loss, specifically through the filtration step. When method optimization is complete, low-template DNA samples could be analyzed recursively without being destroyed during PCR.